Enantioselectivity and residue analysis of cycloxaprid and its metabolite in the pile and fermentation processing of Puer tea by ultraperformance liquid chromatography–high‐resolution mass spectrometry

Abstract The residues of cycloxaprid enantiomers and metabolites are investigated by ultraperformance liquid chromatography–high‐resolution mass spectrometry (UPLC‐HRMS) during raw and ripen Puer tea processing. A Chiralpak AG column with chiral stationary phase of amylose tris (3‐chloro‐5‐methylphenylcarbamate) is succeed to separate the 1R, 2S‐cycloxaprid, 1S, 2R‐cycloxaprid, and their metabolite, which is identified as nitrylene‐imidazolidine. It is not conversed 1R, 2S ‐cycloxaprid into 1S,2R‐cycloxaprid during Puer tea processing. The estimated half‐lives of the 1R,2S‐cycloxaprid and 1S,2R‐cycloxaprid are 0.97 and 1.1 h, respectively, and 1R,2S‐cycloxaprid decreases more quickly than the 1S,2R‐cycloxaprid. During raw Puer tea processing, the half‐lives of 1R, 2S‐cycloxaprid and 1S, 2R‐cycloxaprid are 1.68 h and 1.77 h, but the residue is still detected even if it is over 730 day. However, the half‐lives of 1R,2S ‐cycloxaprid and 1S,2R‐cycloxaprid are 0.60 day and 0.63 day during ripen tea processing. The amounts of metabolite are more in raw tea than in ripen tea; the terminal residues are still detected until 730 days during raw tea. A significant enantioselectivity of 1R, 2S‐cycloxaprid and 1S, 2R‐cycloxaprid is observed during raw tea or ripen tea processing. The degration result shows the enantioselectivity of cycloxaprid in raw or ripen Puer tea processing.


| INTRODUC TION
Neonicotinoids are the most important class of synthetic insecticides for tea protection against piercing-sucking pests (Tomizawa & Casida, 2003). Cycloxaprid is a new neonicotinoid insecticide that has been synthesized and industrialized in China (Li et al., 2011). It is different from traditional neonicotinoids, which act as agonists of native and recombinant nicotinic acetylcholine receptors (nAChRs ) (Liu & Casida, 1993;Matsuda et al., 1998;Nishimura et al., 1994;Tomizawa & Casida, 2003) and show high insecticidal activity against a broad spectrum of sucking and biting insects1 (Cui et al., 2012;Shao et al., 2010), which suggests that it has been considerable as the third generation of neonicotinoids. The molecular structure of cycloxaprid contains a chiral oxabridged cis-configuration leading to a pair of enantiomers, 1R,2S-cycloxaprid and 1S,2R-cycloxaprid ( Figure 1). Cycloxaprids are commonly produced and used as racemic mixtures and the stereoselectivity degrade is found in soils (Liu et al., 2015). Zhang et al. observed stereoselective uptake and translocation of cycloxaprid in edible vegetables from roots (Zhang et al., 2013). However, Chen et al. (Chen et al., 2017) founded adverse results, as evidenced by the lack of significant difference between the stereoisomers in their fate in aerobic soils and three mainly metabolites were found in soil.
The metabolite of cycloxaprid is readily founded through photolysis, hydrolysis, and oxidation reaction. Liu et al. (Liu et al., 2015) identified and tracked 11 metabolites of cycloxaprid, and tracked their changes in flooded and anoxic soils. Shuang et al. (Hou et al., 2017) studied the photostability of cycloxaprid in water and detected 25 photodegradation products; the predominant photodegradation product was named as NTN 32,692. Fang et al. (Fang et al., 2017) reported that the degradation dynamics of two neonicotinoids during Lonicera japonica planting, drying, and tea brewing processes were researched. Hou et al. (Hou et al., 2013) compared the dissipation behavior of three neonicotinoid insecticides in tea and found high transfer rates through green or black tea brewing of 80.5% or 81.6% for thiamethoxam, of 63.1% or 62.2% for imidacloprid, and of 78.3% or 80.6% for acetamiprid. However, the degradation behavior and metabolite of cycloxaprid was still unknown in Puer tea processing.
Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) has also been explored and has shown great potential for untargeted profiling in tea (Gao et al., 2019;Jia et al., 2020). The analysis method of cycloxaprid in tea was scarcely by LC-HRMS. Only Liu et. al. (Liu & Jiang, 2020) reported that the stereoisomer behavior of sulfoxaflor was determined by LC-HRMS during Puer tea and Black tea processing. Therefore, a new analytical method is developed to determine stereoisomer of cycloxaprid and metabolite in Puer tea using LC-HRMS. The method was applied to investigate the stereoselective cycloxaprid degradation during Pu-erh tea processing. The stock solutions were produced by dissolving the cycloxaprid in acetonitrile. All solutions were stored in a refrigerator at -18°C. HPLC-grade acetonitrile and methanol were provided by Tedia Company Inc. The initial dose of cycloxaprid 50 mg/L was used with 1g of cycloxaprid completely dissolved into 5 L water. Water was purified using a Milli-Q system.

| Separation the metabolite of cycloxaprid
One gram of 25% cycloxaprid powder is placed on sun at 3 d. The sample is dissolved by 20 ml water, then extracted by acetonitrile.
The metabolite with degradation test is obtained by semipreparative HPLC. The molecular structure of metabolite is analyzed by LC-HFMS.

| The transform of optical pure compounds in Puer tea processing
The optical pure standards of 1R,2S-cycloxaprid or 1S,2R-cycloxaprid (1 mg/L) are respectively added to research the transform of optical pure compounds in Puer tea processing. The test at intervals time is designed at 0, 2 h, 15 h, 24 h, 48 h, 96 h, and 140 h.

| Degradation in raw Puer tea
Sun-dry Puer tea (20 kg) is obtained and sprayed with 50 mg/L aqueous solution (25% powder) in March, 2019. The raw Puer tea is stored under air temperature (5-28°C) and in dark place. The intervals time is designed at 0 (2 h), 4 h, 10 h, 16 h, 1d, 3d, 6 months, 12 months, 18 months, 24 months, and 36 months. The sample is dried to constant weight and the residues amount calculated with dry sample.

| Ripen Puer tea processing
To ferment the ripen Puer tea, 20 kg of sun-dry Puer teas is sprayed with 25% powder at the dose of 50 mg/L aqueous solution to keep the conditions of 35% moisture content. During the pile-fermentation, the fermented tea was artificially turned and piled again at 7th days

| Calculation of enantiomer fraction
The enantiomer fraction (EF) was EF = R

(R + S)
, concentration of the 1R,2S-cycloxaprid was R, the other concentration of the 1S, 2Rcycloxaprid was S.

| Samples preparation
Five grams of sample was exactly weighed and added 10 ml water, 20 ml acetonitrile. After the mixture was vortexed, 5 g NaCl was added. The tube was shaken vigorously for 1 min using a vortex mixer and centrifuged at 5000 rpm for 5 min. The upper layer solution was mixed with 150 mg PSA and 150 mg anhydrous MgSO 4 for cleanup.
After shaking and centrifugation at 5000 rpm for 3 min, 0.5 ml of the upper layer was filtered through 0.22 μm filter for LC-HFMS analysis.

| UPLC-HRMS Analysis
Sample analysis was achieved in an ultraperformance liquid chromatography-Q exactive high-resolution mass spectrometry (Thermo Fisher Scientific,) system.

| Method validation
The method was validated with the following parameters: matrix effect, accuracy, linear range, limit of detection (LOD), limit of quantification (LOQ), specificity, and precision. The standard solution was determined from 2.0 to 100 μg/ml concentration for each enantiomer. Three times signal-to-noise (S/N) ratio was as the LOD for every enantiomer, whereas the LOQ was based on the lowest spiked concentration level. As shown in

| Chromatography Separation optimized
Because of absence of oxabridged ring, the metabolite was unstereoselective molecule. Once cleavage occurred on the oxabridge, the metabolite is no longer enantioselectivity. To simultaneously separate the chiral cycloxarpid and metabolite, the reverse-phase chiral columns were employed which contained cellulose-and amylosebased polysaccharide materials; a cellulose-based column (Chiral Cel OJ-3R) and two amylose-based columns (Chiralpak AD-RH and Chiralpak IG) were tested using a variety of reverse-phase mobile phase combinations.

| Stereoselective dissipation of cycloxaprid in Puer tea processing
The fermentation processing with under from several months to several ten years is unique to raw Puer tea. So the degradation of cy- Stereoselectivity is expressed as EF value. As shown in Figure 4, the beginning of EF value in cycloxaprid is >0.50, and the decrease of EF is obvious from 2 h to 730 days. The result showed that enantioselectivity is significantly observed during raw Puer tea processing.
The result is shown that the degradation of cycloxaprid is enantioselectivity under raw Puer tea processing.

| Stereoselective dissipation of cycloxaprid in ripen Puer tea processing
Ripen Puer tea is unique processing due to the pile formation at 45 or 60 days. So the degradation of cycloxaprid is detected from starting  The decrease of EF is obvious from 0.56 to 0.44 during ripen Puer tea processing in Figure 6. From the statistical analysis, it showed that there is stereoselective preference for cycloxaprid enantiomers as evidenced as significant difference among the stereoisomers and the racemate in ripen Puer tea processing. The result is shown that the degradation of cycloxaprid is enantioselectivity under raw Puer tea processing.

| Dissipation of metabolite in raw Puer tea processing and ripen Puer tea
When metabolites are produced during Puer tea processing, they are not easy to decompose. So the terminal residue is still detected until 730 days in raw Puer tea processing and 45 days in ripen Puer tea processing (Figure 7). The maximum residue appears at one day (raw Puer tea processing) or earlier (ripen Puer tea processing). The metabolites are higher in residues in raw Puer tea than in ripen Puer tea.

ACK N OWLED G EM ENT
We are grateful for supporting funded National Natural

CO N FLI C T O F I NTE R E S T
All Authors declare that they have no conflict of interest.